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Abstract:

Disclosed is a composition for the inhibition of transplant rejection and
the treatment of skin diseases, comprising a cordyceps mycellia extract
as an active ingredient. The cordyceps mycellia extract significantly
suppresses the production of antibodies to transplants without side
effects, such as weight change. Based on natural material, the
composition is non-toxic and harmless to the human body and thus can be
used as an immunosuppressant for organ transplantation. Also, it stops
oozing from sores and is useful in the treatment of skin diseases,
including atopy, allergic reactions, decubitus ulcers, pemphigus and
smallpox.

Claims:

1. A method for inhibiting transplant rejection comprising administrating
an effective amount of an immunosuppressive composition comprising a
mycelial extract from vegetable worm as an active ingredient to a subject
in need thereof.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application is a continuation application of U.S.
patent application Ser. No. 12/740,139 filed on Apr. 28, 2010, which is a
371 of PCT/KR2007/006356 filed Dec. 7, 2007, the contents of each of
which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to a composition for inhibition of
transplant rejection, comprising a cordyceps mycellia extract as an
active ingredient.

BACKGROUND ART

[0003] Transplant rejection occurs when the immune system of the recipient
of a transplant attacks a transplanted organ or tissue. Thus, effective
suppression of the immune response is known as a main factor determining
the success of transplantation. In this regard, the development of
immunosuppressive medications has brought about exceptional advances in
the transplantation of organs and tissues and the treatment of autoimmune
diseases and has made a great contribution to the study of the in vivo
mechanism of immune responses to the transplanted organ or tissue.

[0004] As described, immunosuppressive drugs were developed to inhibit or
attenuate transplant rejection. An example is cyclosporine A (U.S. Pat.
No. 4,117,118) produced from Tolypocladium inflatum, a soil fungus. These
immunosuppressive drugs not only help realize clinically successful organ
transplantation, but also suggest the therapeutic use thereof in treating
autoimmune diseases. Even though they are required to act selectively and
specifically for T-cells only, conventional immunosuppressive drugs have
an influence on a wide range of cellular functions, including general
signal pathways, causing side effects on other organs, which are healthy
(see. S.-H. Lee et al., Korean J. Immunology, 19:375389 (1997)). For
instance, cyclosporine A is known to show side effects of chronic liver
diseases and hypertension after heart transplantation (see: J. E. F.
Reynolds, et al., Martindale The Extra Pharmacopoeia, 31st ed., pp.
557-562, Royal Pharmaceutical Society, London, 1996). Many attempts have
been made to develop novel immunosuppressive drugs free of side effects.
FK-506 has recently been discovered to be an immunosuppressant, and has
been commercialized. However, side effects of this drug have also been
found (Clin. Transplantation, 11: 237-242 (1997)).

[0005] In China, vegetable worms, together with Korean ginseng, have long
been used as precious materials in medicinal cuisine for special people
in the aristocratic classes. Vegetable worms are a kind of medicinal
fungus produced as a result of the parasitism of vegetable worms on
insects. In high temperature and moisture conditions, the vegetable
worms, which are actually fungus, infect living insects, proliferate
therein to kill the host insects, and form fruiting bodies on the surface
of the host insects. As used herein, the term "vegetable worm", is
intended to primarily refer to Cordyceps sinensis, a parasite on larvae
of the Hepialidae family, but at present generally refers to all fungi
attacking arthropods, such as spiders.

[0006] Cordyceps sinensis breaks down into 10.8% water, 8.4% lipids,
25˜32% crude proteins, 23.9% carbohydrates, and 18.5% crude fibers.
In this vegetable worm are found 17 different amino acids, including 8
essential amino acids. Also, it contains a trace amount of cordycepin,
7.6% of D-mannitol and 11.2% of polysaccharides, all known as medicinally
active materials. Cordycepin, a derivative of the nucleoside adenosine,
is an isomer of quinic acid, known to show anti-cancer activity.

[0008] However, nowhere has the use of vegetable worm extracts as an
immunosuppressant been disclosed in the prior art.

[0009] Leading to the present invention, intensive and thorough research
on an immunosuppressant entailing no side effects, conducted by the
present inventors, resulted in the finding that an extract from vegetable
worm mycelia significantly inhibits the immune response to transplanted
organs or tissues.

DISCLOSURE

Technical Problem

[0010] It is therefore an object of the present invention to provide an
immunosuppressive composition, useful in the prevention of transplant
rejection, comprising a cordyceps mycellia extract as an active
ingredient.

[0011] It is another object of the present invention to provide a
composition for the prevention and treatment of skin diseases, comprising
a cordyceps mycellia extract as an active ingredient.

Technical Solution

[0012] In order to accomplish the objects of the present invention, an
immunosuppressive composition comprising a mycelial extract from a
vegetable worm as an active ingredient is provided for the inhibition of
transplant injection.

[0013] Also, a composition comprising a cordyceps mycellia extract as an
active ingredient is provided for the prevention and treatment of skin
diseases.

Advantageous Effects

[0014] The cordyceps mycellia extract was found to significantly suppress
the production of antibodies to transplants without side effects, such as
weight change. Based on a natural material, the composition is non-toxic
and harmless to the human body, and thus can be used as an
immunosuppressant for organ transplantation. Also, it arrests oozing from
sores and is applicable to the prevention and treatment of skin diseases,
including atopy, allergic reactions, decubitus ulcers, pemphigus and
smallpox.

DESCRIPTION OF DRAWINGS

[0015] FIG. 1 is a graph showing the levels of antibodies produced against
splenocytes transplanted into mice administered with the cordyceps
mycellia extract of the present invention or with saline in accordance
with an embodiment of the present invention (.box-solid.: control,
Δ: experimental group).

[0016]FIG. 2 is a graph showing changes in the weight of the mice
administered with the cordyceps mycellia extract of the present invention
and with saline in accordance with an embodiment of the present invention
(.box-solid.: control, Δ: experimental group).

[0017] FIG. 3 shows the dermal states of mice administered with the
cordyceps mycellia extract of the present invention and with saline in
photographs (panel a: experimental group, panel b: control).

[0018] FIG. 4 is a graph showing the levels of antibodies produced against
splenocytes transplanted into mice administered with the cordyceps
mycellia extract of the present invention or with other comparative
chemicals in accordance with another embodiment of the present invention.

[0019] FIG. 5 is a graph showing changes in the weight of the mice
administered with the cordyceps mycellia extract of the present invention
and with other comparative chemicals in accordance with another
embodiment of the present invention.

BEST MODE

[0020] In accordance with an aspect of the present invention, an
immunosuppressive composition based on a cordyceps mycellia extract is
provided for the inhibition of transplant rejection.

[0021] The cordyceps mycellia extract may be obtained from cultured
mycelia, or may be commercially available. For example, vegetable worm
powder, which is sold as a health aid food, may be used in the present
invention, whether it comes from fruit bodies, mycelia, or a combination
thereof.

[0023] The cordyceps mycellia extract according to the present invention
was tested for immunosuppressive effect on smallpox mouse models. After
the transplantation of splenocytes thereinto, the animal models were
administered with the cordyceps mycellia extract. ELISA analysis on the
animal models for the quantification of antibodies to the splenocytes
indicated that the cordyceps mycellia extract of the present invention
significantly inhibits the production of the antibodies (see FIGS. 1 and
4). Therefore, the cordyceps mycellia extract according to the present
invention can be used as an immunosuppressant for the prevention of
transplant rejection.

[0024] Also, observations were made of whether the cordyceps mycellia
extract causes side effects in vivo. Almost no changes were found in the
weight of the mice after the transplantation of splenocytes (see FIGS. 2
and 5). Hence, the cordyceps mycellia extract can be used as an
immunosuppressant for the prevention of transplant rejection without the
occurrence of side effects, such as changes in weight, upon organ
transplantation.

[0025] In accordance with another aspect thereof, the present invention
provides a composition for the prevention and treatment of skin diseases,
comprising a cordyceps mycellia extract as an active ingredient.

[0026] The cordyceps mycellia extract according to the present invention
was tested for therapeutic activity for skin diseases on smallpox mouse
models. After the administration of the cordyceps mycellia extract
thereinto, the smallpox mouse models were observed to stop oozing from
the smallpox sores (FIG. 3). In contrast, the control, which was not
administered with the cordyceps mycellia extract of the present
invention, was observed to ooze from the smallpox sores and remain
depilated at the sores. Hence, the cordyceps mycellia extract according
to the present invention can be used for the treatment of skin diseases,
such as atopy, allergic reactions, decubitus ulcers, pemphigus, smallpox,
etc.

[0027] When used as medications or health aid foods, the composition may
further comprise one or more active ingredients having a function similar
to that of the cordyceps mycellia extract.

[0028] The cordyceps mycellia extract in accordance with the present
invention can be administered orally or non-orally, and may be provided
in general medicinal forms. For clinical practice, the cordyceps mycellia
extract of the present invention may be used in oral or non-oral forms.
It is usually formulated in combination with a diluent or an excipient,
such as a filler, a thickening agent, a binder, a wetting agent, a
disintegrant, a surfactant, etc. Solid agents intended for oral
administration of the extract of the present invention may be in the form
of tablets, pills, powders, granules, capsules, and the like. In these
solid agents, the cordyceps mycellia extract of the present invention is
formulated in combination with at least one excipient, such as dextrin,
starch, calcium carbonate, sucrose, lactose, or gelatine. In addition, a
lubricant, such as magnesium stearate, talc, or the like, may also be
added. Liquid agents intended for oral administration include
suspensions, internal use solutions, emulsion, syrups, and the like. In
addition to a simple diluent, such as water or liquid paraffin, various
excipients, such as wetting agents, sweetening agents, aromatics,
preservatives, and the like, may be contained in the liquid agents for
the oral administration of the extract of the present invention. Also,
non-oral dosage forms of the extract of the present invention include
sterile aqueous solutions, non-aqueous solutions, suspensions and
emulsions, freeze-dried agents, and suppositories. Non-aqueous solutions
and suspensions made from propylene glycol, polyethylene glycol,
vegetable oils, such as olive oil, and injectable esters such as ethyl
oleate may be used. The basic materials of suppositories include
Witepsol, macrogol, Tween 61, cacao butter, laurin, glycerol, and
gelatin.

[0029] The effective dosage of the cordyceps mycellia extract in
accordance with the present invention depends on various factors,
including the patient's weight, age, gender, state of health, diet, the
time of administration, route of administration, excretion rate, etc. For
oral administration, the cordyceps mycellia extract in accordance with
the present invention may be administered at a dose ranging from 550 to
2,200 mg/day.

[0030] For application to the treatment of skin diseases, the cordyceps
mycellia extract of the present invention may be used alone or in
combination with other therapies, including surgery, radiotherapy,
hormonal therapy, chemical therapy and/or biological reaction regulators.

MODE FOR INVENTION

[0031] A better understanding of the present invention may be obtained in
light of the following examples, which are set forth to illustrate, but
are not to be construed to limit the present invention.

Example 1

Immunosuppression Assay on Smallpox Mouse Model

[0032] An immunosuppression test was conducted with the cordyceps mycellia
extract of the present invention on smallpox mouse models (obtained from
Microbiology•Immunology Lab of the Medical College in Keio Univ.).

[0033] From two days before the transplantation of splenocytes (Dsg3-/-),
five smallpox mice were administered orally with a cordyceps mycellia
extract powder (Cordyma®; Han Kook Sin Yak) at a dose of 10
mg/kg/day. For 35 days (5 weeks) after the splenocyte transplantation,
the cordyceps mycellia extract was orally administered at a dose of 10
mg/kg/day. For a control, physiological saline was used instead of the
extract.

[0034] Blood samples were taken from the mice 7 days (1 week), 14 days (2
weeks), 21 days (3 weeks), 28 days (4 weeks) and 35 days (5 weeks) after
the transplantation, and were analyzed using ELISA to determine the level
of antibodies to the splenocytes. Throughout the experiment, the weights
and skin states of the mice were monitored every day.

[0035] The results are depicted in FIGS. 1 to 3.

[0036] In FIG. 1, the levels of antibodies to the splenocytes are plotted
against time for the experimental group and the control group. FIG. 2
shows changes in weight for the experimental group and the control group.
In FIG. 3, the experimental group and the control group are compared with
each other with respect to skin state.

[0037] As seen in FIG. 1, the level of antibodies to the transplanted
splenocytes in the mice administered with the cordyceps mycellia extract
of the present invention was almost zero. From this, it is apparent that
the cordyceps mycellia extract according to the present invention
effectively inhibits the production of antibodies to transplants.

[0038] In FIG. 2, it can be observed that the weight of the control
administered with physiological saline slightly increased immediately
after the transplantation, but sharply decreased from 7 days after the
transplantation, as transplant rejection occurred. In contrast, almost no
change was found in the weight of the mice administered with the
cordyceps mycellia extract of the present invention. Demonstrated to
effectively inhibit the transplant rejection and cause no side effects,
such as weight gain, the composition comprising the cordyceps mycellia
extract according to the present invention can therefore be used as an
immunosuppressive medication applicable for organ or tissue
transplantation.

[0039] Furthermore, as shown in FIG. 3, while the control suffered from
sores due to smallpox, skin disease was suppressed in the mice
administered with the cordyceps mycellia extract of the present
invention. Hence, the cordyceps mycellia extract according to the present
invention can be applied to the treatment of skin diseases including
atopy, allergic reactions, decubitus ulcers, pemphigus, smallpox, etc.

Example 2

Immunosuppression Assay on Pemphigus Mouse Model

[0040] An immunosuppression test was conducted with the cordyceps mycellia
extract of the present invention on pemphigus mouse models, as follows.

[0041] Two S129 Dsg3-/- mice and 24 S129 Rag2-/- mice were prepared as
transplantation donors and recipients, respectively. The recipient mice
were divided into four groups of six: control (CMC administered),
comparative group (cyclophosphamide (CPA) administered), experimental
group 1 and experimental group 2 (cordyceps mycellia extract
(Cordyma®; Han Kook Sin Yak) administered). The transplantation donor
was subjected to an immune reaction by administering a Dsg3-His protein
thereto, as described below. First, 10 μg of mouse Dsg3-His protein
was emulsified with the same amount of complete Freund's adjuvant (CFA)
and subcutaneously injected into the mice (1st). One week after the
first immunization, an emulsion of 10 μg of mouse Dsg3-His protein in
the same amount of incomplete Freund's adjuvant was subcutaneously
injected (2nd). One week after the 2nd immunization,
subcutaneous injection was carried out in the same manner as in the
second immunization (3rd). One week later, 10 μg of the protein
was intraperitoneally administered (4th) One week after the 4th
immunization, intraperitoneal injection was performed in the same manner
as in the 4th immunization (5th). Three days before
transplantation, intraperitoneal injection was performed in the same
manner as in the 4th immunization.

[0042] From one day before the transplantation of splenocytes (Dsg3-/-),
oral administration was conducted with 1 ml of the cordyceps mycellia
extract (Cordyma®; Han Kook Sin Yak) for each experimental group, 1
ml of CMC for the control group and 1 ml of cyclophosphamide for the
comparative group. Thereafter, splenocytes were transplanted at a density
of 1.5×106 cells/500 μl into each mouse, followed by oral
administration of 1 ml of the test material to the mice on day zero, 1,
4, 7, 11, 14, 18, 21, 25, and 28 after the transplantation. Afterwards,
blood samples were taken from the mice on day zero, 1, 4, 7, 11, 14, 18,
21, 25 and 28 after the transplantation, and were analyzed to determine
the level of antibodies to the splenocytes using ELISA. Throughout the
experiment, the weights and skin states of the mice were monitored every
day.

[0043] The cordyceps mycellia extract (Cordyma®; Han Kook Sin Yak) for
the experimental group 1 was prepared by placing 500 mg of a powder of
Isaria japonica in a 15 ml tube, adding a 0.5% CM-Na solution
(hereinafter referred to as "CMC") to the tube to form a total volume of
5 ml, and sonicating it for 30 min in a bath to form a suspension
(dosage: 2000 mg/kg/administration). As for the experimental group 2, its
cordyceps mycellia extract (Cordyma®; Han Kook Sin Yak) was prepared
by mixing 0.5 ml of the cordyceps mycellia extract (Cordyma®; Han
Kook Sin Yak) for the experimental group 1 with 4.5 ml of CMC and
treating the mixture for 30 min in a sonication bath to give a suspension
(dosage: 200 mg/kg/administration).

[0044] Also, the cyclophosphamide administered to the comparative group
was a suspension prepared by pulverizing 50 mg of the Endoxan P tablet,
commercially available from Shionogi Pharmaceutical Corporation, mixing
the powder with 16.7 ml of CMC, and sonicating the mixture in a bath for
30 min (dosage: 60 mg/kg/administration).

[0045] Their dermal conditions were examined on the abdominal side, the
dorsal side, the right side, and the left side for blistering, depilation
and swelling with the naked eyes while pictures were taken of the entire
dorsal side and three facial sides (right, left and chin) with a digital
camera for more precise monitoring.

[0046] The results are shown in FIGS. 4 and 5.

[0047] In FIG. 4, the levels of the antibodies to the splenocytes were
plotted against time for the experimental groups, the control group and
the comparative group. FIG. 5 shows changes in weight for the
experimental groups, the control group and the comparative group.

[0048] As seen in FIG. 4, the production level of antibodies to the
transplanted splenocytes was observed to be lower in the mice
administered with the cordyceps mycellia extract of the present invention
than in the mice administered with CMC only (control). As for ELISA
values, a Dunnett's multiple comparison test was performed on the basis
of a PBS group. On Day 11 after the transplantation, there was
significance for the experimental group 1 (p<0.05), but not
significance for the experimental group 2. On Day 14, there was
significance for both the experimental group 1 (p<0.01) and the
experimental group 2 (p<0.05). However, no significance was observed
for either of them from Day 18. Accordingly, the significant difference
on Days 11 and 14 between the experimental group administered with the
cordyceps mycellia extract at a dose of 2000 mg/kg and the control group
indicates that the cordyceps mycellia extract of the present invention
significantly inhibits the production of antibodies responsible for
transplant rejection.

[0049] For weight change, a Dunnett's multiple comparison test was
performed on the basis of the control group. On Day 11 after the
transplantation, as shown in FIG. 5, there was significance for the
experimental group 1 (p<0.05), but not significance for the
experimental group 2. On Day 14, there was significance for both the
experimental group 1 (p<0.05) and the experimental group 2
(p<0.05). However, no significance was observed for either of them
from Day 18. Accordingly, these results indicate that the cordyceps
mycellia extract of the present invention was found to effectively
suppress transplant rejection, as demonstrated by the significant
difference on Days 11 and 14 between the experimental group administered
with the cordyceps mycellia extract at a dose of 2000 mg/kg and the
control group.

[0050] Furthermore, while the control suffered from skin diseases, such as
sores and depilation due to pemphigus, the skin diseases were suppressed
in the mice administered with the cordyceps mycellia extract of the
present invention. Hence, the cordyceps mycellia extract according to the
present invention can be applied to the treatment of skin diseases
including atopy, allergic reactions, decubitus ulcers, pemphigus,
smallpox, etc.

[0051] The composition of the present invention can be prepared as
described below.

Preparation Example 1

Preparation of Pharmaceutical Formulations

[0052] 1-1. Preparation of Powder

TABLE-US-00001
Cordyceps mycellia extract 1 g
Dextrin 0.1 g

[0053] The above ingredients were mixed and loaded into an airtight sac to
produce powder.

[0059] The cordyceps mycellia extract was dissolved in a suitable volume
of an NaCl BP injection, and the solution was adjusted to a pH of 3.5
with diluted HCl BP and to a desired volume with NaCl BP injection,
followed by sufficient mixing. The solution was loaded into transparent 5
ml type I ampules, which were hermetically sealed by melting, followed by
autoclaving at 120° C. for 15 min to prepare injections.